Project Summary/Abstract Fluorinated pharmaceuticals represent a rapidly expanding class of small molecule drugs that have become important in the treatment of diverse human health conditions ranging from cancer to high cholesterol. Indeed, the selective introduction of fluorine into compounds has become a key strategy for chemists to rationally tune the behavior of small molecules in order to turn a lead compounds into an effective treatment. Indeed, ~30% of pharmaceuticals now contain fluorine, including three of the top five-selling drugs. However, these same chemical properties limit our ability to specifically incorporate fluorine into molecules and consequently our ability to fully exploit the potential that fluorine provides as a design element in the pipeline of drug design and discovery. New developments in synthetic chemical methods have greatly improved our ability to make carbon-fluorine bonds, but challenges in the synthesis and preparation of organofluorine compounds continue to restrict the scope of molecular structures and methods that can be used to screen for new drug candidates. Our group has been developing alternative synthetic biology approaches to engineer enzymatic and living systems for the production of complex fluorinated compounds with bioactivity, with the long-term goal of developing new approaches to fluorinated drug discovery. Specific aims of this proposal include: (i) elucidating the molecular mechanism of naturally-occurring fluorine selectivity in enzymes from Streptomyces cattleya, one of the few known native organofluorine-producing organisms, in order to build a knowledgebase for engineering fluorine-selective enzymes, (ii) studying the mechanism of fluorinated extender unit usage in polyketide synthases, which produce a large family of medicinally-important natural products, and (iii) developing in vitro and in vivo methods for production of fluorinated natural products in the polyketide family.